Our objective is to develop a method for producing unique and known base sequence changes at random sites in the DNA of any gene in vitro and to use the method to investigate the plasmid pBR322. A small number of base pairs will be deleted from random sites on the plasmid prior to the addition of commercially synthesized 4 or 6 base-pair oligomers containing recognition sites for restriction endonucleases MboI, XbaI or HpaI. For a significant fraction of the molecules there will be an exchange of 4 or 6 base pairs. The 4 and 6 nucleotide exchanges will lead to single amino acid changes in 43 percent and 10 percent of the cases, respectively. This type of nucleotide exchange is expected to produce temperature-sensitive (ts) mutations. A large number of these ts mutants will be isolated in the ampicilin (AP) and tetracycline (Tc) resistance genes on pBR322. Some ts mutants will be remutagenized by the same procedure to isolate temperature-independent revertants. Nonsense mutants, produced by mutagenesis of pBR322 DNA, will be screened by constructing a pBR322-ColEl hybrid plasmid and using F-mediated transfer of the hybrid plasmid into recipient cells containing suppressors of nonsense codons. A minicell protein synthesis system will be used to identify the truncated proteins made by nonsense mutants. All mutants produced by the nucleotide exchange mechanism will be mapped on pBR322 using the new restriction sites created by mutagenesis. Restriction-site mapping will provide valuable information about the positions and control of expression of Tcr genes as well as the structure and processing of beta-lactamase protein. DNA sequencing will be done for a few of these mutants to confirm that they contain the expected base substitutions.